This invention relates generally to identifying molecules in a polymer chain, and more particularly to sequencing such molecules through tunneling current measurements.
A great deal of research has been performed relating to the functionality of biopolymers, which is determined by the primary sequence of the monomers within the biopolymer. Identifying the sequence of monomers is integral to the understanding of the functionality of the biopolymer. Rapid, reliable, and inexpensive characterization of polymers, particularly nucleic acids, has become increasingly important. Typical current nucleic acid sequencing methods depend either on chemical reactions that yield multiple length DNA fragments cleaved at specific bases, or on enzymatic reactions that yield multiple length DNA fragments terminated at specific bases.
Each of the known methods for sequencing polymers has drawbacks. For instance most of the methods are slow and labor intensive. The gel based DNA sequencing methods require approximately 1 to 3 days to identify the sequence of 300-800 bases in length. Methods such as mass spectroscopy and ELIDA sequencing can only be performed on very short polymers.
Recently, the development of natural or man-made nanopores has enabled rapid determination of the sequence of nucleic acid molecules. In nanopore sequencing, single stranded DNA is passed through a nanopore in a suitable solution and individual nucleotides (or physical changes in the environment of the nucleotide) are physically sensed. For example, a membrane with a nanopore separates two chambers in a solution, between which a low voltage is applied. The ionic current in the solution between the two chambers via the nanopore is used to monitor the presence of the DNA inside the nanopore. When a single stranded DNA is in the nanopore, it partially blocks the nanopore so that the ionic current between the two chambers is decreased. It is proposed to use the change of the ionic current to identify the DNA bases. (See, for example, “Rapid nanopore discrimination between single polynucleotide molecules”, Proc. Natl. Acad. Sci. USA. 97:1079-85, 2000; Baldarelli et al., U.S. Pat. No. 6,015,714; and Church et al., U.S. Pat. No. 5,795,782, each incorporated herein by reference.) Since there are typically approximately ten DNA bases in the nanopore at any given time due to the aspect ratio of the nanopore, using ionic current change to identify the individual DNA base is very difficult.
On the other hand, the development in nano-technology makes it feasible to limit the passage of molecules through nanopores. For example, the use of membrane channels to characterize polynucleotides as the molecules pass through the nanopores has been studied. Kasianowicz et al. (Proc. Natl. Acad. Sci. USA. 93:13770-3, 1996, incorporated herein by reference) used an electric field to force single stranded RNA and DNA molecules through a 2.6 nanometer diameter ion channel in a lipid bilayer membrane. The diameter of the channel permitted only a single strand of a nucleic acid polymer to traverse the channel at any given time. As the nucleic acid polymer traversed the channel, the polymer partially blocked the channel, resulting in a transient decrease of ionic current. Since the duration of the decrease in current is directly proportional to the length of the nucleic acid polymer, Kasianowicz et al. (supra) were able to determine experimentally lengths of nucleic acids by measuring changes in the ionic current.
A continuing need exists in the art for the identification and/or sequencing of polymers such as biomolecules that have not previously been identified or characterized.
Described below are improved methods and apparatus for direct sensing or identification of molecules.